The parathyroid hormone (PTH) is formed in the parathyroid gland (Glandulae parathyroideae) and secreted into the blood circulation. In the intact form it consists of a single polypeptide chain having 84 amino acids and a molecular weight of ca. 9500 Dalton (see SWISS-PROT:P01270, PTHY-HUMAN showing the amino acid sequence MIPAKDMAKV MIVMLAICFL TKSDGKSVKK RSVSEIQLMH NLGKHLNSME RVEWLRKKLQ DVHNFVALGA PLAPRDAGSQ RPRKKEDNVL VESHEKSLGE ADKADVNVLT KAKSQ) (SEQ ID NO:1). Together with vitamin-D and calcitonin it brings about the mobilization of calcium and phosphate out of the bone skeleton and increases the uptake of calcium in the intestines and the excretion of phosphate via the kidneys. The concentration of biologically active PTH peptides in plasma or serum is thus an important diagnostic parameter for determining presence and degree of hyper- or hypo-parathyroidism; for a quantification of osteoblast and/or osteoclast activity; a treatment with vitamin-D and vitamin-D metabolites; an estimation of the presence of aluminum or a possible oestrogen deficiency in post-menopausal dialysis patients; for determining the steroid or cyclosporine dosage after kidney transplantations or a treatment or prevention of pathological bone marrow changes, uraemic conditions and chronic kidney failure.
Secondary hyper-parathyroidism further occurs frequently in chronic kidney disease as an adaptive response to deteriorating renal function. This is because circulating 1,25-dihydroxy vitamin D starts to decrease very early in stage 2 of chronic kidney disease and continues to fall as the glomerular filtration rate (GFR) decreases further, and the renal 1α-hydroxylase is inhibited by hyperphosphatemia, hyper-uricemia, metabolic acidosis as well as 25-hydroxyvitamin D deficiency. As GFR decreases below 60 mL/min/1.73 m2 phosphate is retained which stimulates secretion of PTH. Hypocalcaemia develops as the GFR decreases below 50 mL/min/1.73 m2, further stimulating a release of PTH. With disease progression, intact PTH (aa 1-84) half-life increases and C-terminal fragments of the hormone accumulate in serum. A relative state of end-organ resistance to the hormone exists but chronic elevation of it has major consequences resulting in bone loss, particularly of cortical bone, fractures, vascular calcification, cardiovascular disease, and hence an increased cardiovascular mortality (cf Fraser W D, Hyperparathyroidism. Lancet 2009; 374:145f) A reliable method of determining the concentration of biologically active PTH peptides in serum is therefore key for detecting patients with hyperparathyroidism as well as for subsequent monitoring of therapeutic interventions.
The first generation of immunoassays for measuring PTH in serum were based on radiolabeled bovine PTH peptides and polyclonal antisera against parathyroid hormone (Berson S A et al. Proc Natl Acad Sci USA. 1963; 49:613-617). As the biologic activity is located in the amino-terminal portion of the PTH peptide and the PTH peptide following its secretion into circulation degraded within minutes in active and inactive fragments, the radioimmunoassay were also detecting inactive degradation products. The first generation of PTH assays therefore produced no reliable clinical measurements since the sera of patients with a renal failure contain high concentrations of inactive PTH fragments.
The second generation of immunoassays uses two antibodies, one binding in the amino-terminal portion of the PTH peptide with the biologic activity and the other in its C-terminal portion. The characterising with synthetic fragments showed however that these immunoassays also determined an inactive large PTH (aa 7-84) fragment (John M R et al. (1999), J. Clin. Endocrinol. Metab., 84. 4287-4290; Gao P et al. 2000, Poster M455, ASBMR 22nd Annual Meeting; Roth H J et al. (2000), Poster P1288; 11th International Congress of Endocrinology, Sydney). This co-determination of the inactive large PTH fragment (7-84) was made responsible for the discrepancy between measured PTH concentrations and clinical findings as the large PTH fragment is likely competing with intact PTH peptides for the binding site of the PTH receptor.
A third generation PTH assay has been developed to overcome the problems with inactive large PTH fragments, which however fails to improve the diagnosis of bone diseases or other clinical signs of secondary hyperparathyroidism in uraemic patients (Brossard J H et al., Influence of glomerular filtration rate on non-(1-84) parathyroid hormone (PTH) detected by intact PTH assays, Clin Chem. 2000; 46:697-703). There have been speculations about systematic errors in the determination or a PTH resistance of osteoblasts or a genetically reduced expression of PTH receptor.
In summary, it is generally accepted in the field that the parathyroid hormone is cleaved in liver, kidney and circulation within minutes into active and inactive fragments and that some fragments have a biological activity comparable with intact PTH peptides whereas others such as hPTH (3-34) seem to inhibit the effects of parathyroid hormone (see EP-A 0 349 545; Schmidt-Gayk et al. (1999) Osteologie forum, 5, 48-58), Suva et al. (1987) Science, 237, 893ff; EP 0 451 867). Moreover, that large PTH non-(1-84) fragments may lead to erroneous determinations (LePage R. et al. (1998) Clin. Chem., 44, 805-809). The term “large PTH fragment” has been coined for PTH fragments which lack amino acid residues at the amino-terminus but which are detected by 2nd generation PTH assays. Additionally, dipeptidyl peptidase-4 (DPP4) is expressed on the surface of many cell types and a rather indiscriminate serine exopeptidase. This led to the hypothesis of PTH further being in vivo a substrate of DPP4 or a similar exoproteinase. Consequently, a two-site immunoassay has been developed employing antibodies that can distinguish between biologically active and biologically inactive PTH peptides that are missing the utmost 2 amino-terminal amino acids (see WO 2001/44818 (Armbruster et al), WO 96/10041 (Mageriein et al); WO 2003/03986 (Hutchison J S)).
However, it was found that serum samples of uraemic patients may contain intact PTH polypeptide chains which are inactive because oxidized at one of its methionines. Such kind of oxidation seems to be particularly relevant for dialysis patients whose blood plasma is exposed to oxidative stress. This led to the development of an immunoassay for determination of non-oxidized PTH (aa 1-84) and biologically active fragments thereof (WO 2002/082092). Notwithstanding, it needs to be ascertained why uraemic patients with normal bone transformation sometimes have serum levels of intact PTH which are more than 2.5 higher than in patients with healthy kidneys (pathological limit in the case of patients with healthy kidneys: 65 μg PTH/L; for patients having uraemic conditions: 165 μg PTH/L serum). Further, uraemic patients with relatively high PTH values often manifest significant differences in bone transformation (Slatopolsky E et al. (2000), Kidney Int., 58, 753-761). Thus these patients often have in the serum eight to ten times increased PTH concentrations, but low normal values for bone specific alkaline phosphatase (ostase). These patients seem to free from symptoms of an excessive PTH activity.
The state of the art therefore still represents a problem. It is further an object of the invention to make available a fast and reliable method for the determination of active parathyroid hormone in a sample of a body fluid, which method particularly allows an early detection of a deteriorating renal function.